It has been established for more than a decade that gene transcription can be initiated within minutes after the activation of cell surface receptors by polypeptide ligands (reviewed in [Levy, D. E. and Darnell, J. E., New Biologist 2: 923-928 (1990)] and Darnell, J. E., Proc. Natl. Acad. Sci. (USA), 94:11767-11769 (1997)]. One of the most direct pathways of polypeptide stimulated gene activity is the so-called Jak-STAT pathway [Briscoe et al., Phil Trans. Royal Soc. (London) B351: 167-171 (1996); [Darnell, 1997; Ihle et al., Annu. Rev. Immunol., 13:369-398 (1995); Leaman et al., FASEB J., 10:1578-1588 (1996)]. STATs are so named because they serve both as signal transducers in the cytoplasm and activators of transcription in the nucleus. Each STAT molecule contains a Src-homology 2 (SH2) domain, a modular unit that binds specifically to phosphotyrosine [Kuriyan, J. and Cowburn, D., Annul. Rev. Biophys. Biomol. Struct. 26:259-288 (1997); Pawson, T., Nature, 373:573-580 (1995)]. The STAT SH2 domain acts as a phosphorylation-dependent switch that controls receptor recognition and DNA binding, thus allowing the STATs to couple the activation of cell surface receptors to gene regulation in a direct manner [Darnell, J. E., Proc. Natl. Acad. Sci. (USA), 94:11767-11769 (1997)].
In animal cells, activation of the latent cytoplasmic STAT molecule is accomplished either through cell surface receptors for cytokines and their non-covalently associated Jak kinases, or by growth factor receptors with intrinsic tyrosine kinase activity [Ihle et al., Annu. Rev. Immunol., 13:369-398 (1995)]. Binding of the cognate ligand to the cell surface receptor causes the phosphorylation of tyrosines in the cytoplasmic regions of the receptor, thus creating docking sites for the STAT SH2 domain. The consequent recruitment of the STATs to the receptor leads, in turn, to their phosphorylation on tyrosine by the Jak or receptor kinases. The phosphorylated STATs form SH2-mediated dimers and are then translocated to the nucleus, where they bind to DNA and direct specific transcriptional initiation [Darnell, J. E., Proc. Natl. Acad. Sci. (USA), 94:11767-11769 (1997)]. STAT-1 and STAT-2 were originally discovered as transcription factors that are activated by interferons .alpha. and .gamma. [Fu, X.-Y. et al., Proc. Natl. Acad. Sci. (USA), 87:8555-8559 (1990); Fu, X.-Y et al., Proc. Natl. Acad. Sci. (USA) 89:7840-7843 (1992); Schindler, C. et al., Proc. Natl. Acad. Sci. (USA) 89:7836-7839 (1992); Veals, S. A. et al., Mol. Cell Biol., 12:3315-3324 (1992)]. Seven mammalian STAT proteins have been discovered so far, and over 40 different polypeptides are now known to activate one or more STATs [reviewed in Darnell, J. E., Proc. Natl. Acad. Sci. (USA), 94:11767-11769 (1997)].
Several U.S. patents and pending U.S. patent applications describe structural features and functions of STAT proteins including, U.S. Pat. No. 5,716,622, and pending patent applications Ser. Nos: 08/820,754, filed Mar. 19, 1997; 08/951,130 filed Oct. 15, 1997; 09/012,710 filed Jan. 23, 1998, all of which are hearby incorporated by reference in their entireties. However, further efforts at dissecting the STATs into separable domains with distinct functions such as DNA binding have met with limited success. Molecular genetic experiments have, however, implicated specific regions of the protein in specific functions. A single phosphorylation site at Tyr 701 of STAT-1 was identified, and proven to be necessary for STAT activity [Shuai, K. et al., Nature 366:580-583 (1993)]. Just upstream from this residue is an SH2 domain, and biochemical experiments indicate that the SH2 domain and the phosphotyrosine in each of two STATs interact in a reciprocal manner to form a dimer [Shuai, K. et al., Cell 76:821-828 (1994)]. The potential DNA binding region of the STATs was shown to include residues in the 400-500 region [Horvath, C. M. et al., Genes Dev. 9:984-994 (1995); Schindler, U. et al., Immunity 2:689-697 (1995)]. However, the architecture and mechanism of this DNA binding region has not been fully elucidated.
Regions of STAT that are upstream from the DNA binding region appear to be involved in protein--protein interactions. An IRF family member, p48, has been shown to interact with a region around Lys 161 in the ISGF3 protein complex [Horvath, C. M. et al., Mol. Cell. Biol. 16:6957-6964 (1996); Martinez-Moczygemba, M. et al., J. Biol. Chem. 272:20070-20076 (1997)]. Furthermore, CBP interacts with the N-terminal 150 residues [Zhang, J. J. et al., Proc. Natl. Acad. Sci. 93:15092-15096 (1996)]. The amino-terminal 130 residues form a separable functional domain (N-Domain) that strengthens interactions between STAT dimers on adjacent DNA binding sites [Vinkemeier, U. et al., EMBO J. 15:5616-5626 (1996); Vinkemeier, U. et al., Science 279:1048-1052 (1998); Xu, X. et al., Science 273:794-797 (1996)].
A deeper understanding of the mechanism of transcriptional activation by the STATs and the role of tyrosine phosphorylation in controlling this activity is impeded greatly by the lack of three-dimensional structural information. Therefore, there is a need to obtain agonists and antagonists that can modulate the effect of STAT proteins during specific gene activation. In particular, there is a need to obtain drugs that will directly interact with the core portion of STAT proteins. Unfortunately, identification of such drugs have heretofore relied on serendipity and/or systematic screening of large numbers of natural and synthetic compounds. A far superior method of drug-screening relies on structure based drug design. In this case, the three dimensional structure of a protein or protein fragment is determined and potential agonists and/or potential antagonists are designed with the aid of computer modeling [Bugg et al., Scientific American, Dec. 92-98 (1993); West et al., TIPS, 16:67-74 (1995)]. However, heretofore the three-dimensional structure of a STAT protein or fragment thereof has remained unknown, essentially because no such protein crystals had been produced of sufficient quality to allow the required X-ray crystallographic data to be obtained.
Therefore, there is presently a need for obtaining a fragment of the core portion of the STAT protein that can be crystallized to form a crystal with sufficient quality to allow such crystallographic data to be obtained. Further, there is a need for such crystals. Furthermore there is a need for the determination of the three-dimensional structure of such crystals. Finally, there is a need for procedures for related structural based drug design based on such crystallographic data.
The citation of any reference herein should not be construed as an admission that such reference is available as "Prior Art" to the instant application.